The present invention relates to a high-purity von Willebrand factor (vWF) preparation as well as to a process for making such preparation. The invention further relates to the use of such preparation for the treatment of bleeding disorders and to a pharmaceutical composition comprising such preparation and suitable for treating these disorders. In a further aspect, the present invention relates to a method for treating bleeding disorders. The bleeding disorder may suitably be von Willebrand""s Disease (vWD), haemophilia A induced by vWF-dependent FVIII deficiency or haemophilia A not induced by such deficiency.
vWF is an adhesive glycoprotein synthesised by megakaryocytes and endothelial cells. vWF circulates in plasma as an array of multimers of increasing size, from. the protomers of approximately 500 kD to the largest multimers having a molecular weight of approximately 20,000 kD. Each protomer of 500 kD is made up of two identical monomeric subunits and is therefore also referred to as dimer. The multimers are made up of repeating units of protomers. vWF plays an important role in normal haemostasis due to two main functions. Firstly, vWF is essential to coagulation due to its ability to serve as a carrier of coagulation factor VIII (FVIII), which is a plasma protein that participates in the intrinsic pathway of blood coagulation. The formation of such complexes protects FVIII from proteolytic inactivation. Secondly, vWF contributes, through different mechanisms, to platelet adhesion and aggregation.
Disturbances in blood plasma level of vWF and/or FVIII lead to very serious, sometimes life-threatening, bleeding disorders.
von Willebrand""s Disease (vWD) is an autosomal inherited bleeding disorder, the onset of which is due to deficiency and/or abnormality of vWF. As a consequence, FVIII coagulant activity (FVIII:C) is often reduced, since, as mentioned above, vWF serves as a carrier of FVIII. The relevance of vWF for platelet adhesion to the damaged endothelium is reflected by the fact that patients with vWD also often have prolonged skin bleeding time.
vWD is the most common inherited bleeding disorder. It has been demonstrated that its prevalence in the general population is at least 0.8%, and a multiethnic paediatric survey (made in 1993) in the US has given a figure of 1.3%, confirming that the disease has no racial differences and is distributed world-wide.
Epistaxis, bleeding after dental extraction and menorrhagia are the most prominent symptoms, being present in about 60% of the affected patients. Often, bleeding symptoms lessen with age in patients with a mild form of the disease. Epistaxis is very frequent during childhood. Menorrhagia is the most frequent clinical problem in women with vWD, often requiring the combination of oral contraceptive pill and iron supplementation. In patients with classical vWF deficiencies, postpartum bleeding is not often encountered since vWF level usually increases during pregnancy, whereas patients with the variant forms usually require peripartum replacement therapy. Patients with severe disease may experience muscular or joint bleeding because of the concomitant markedly reduced FVIII levels. Furthermore, bleeding after surgery may be life-threatening.
Three main types of vWD have been identified. Type 1, with classical autosomal dominant inheritance, accounting for about 70% to 80% of cases, is characterised by equally low FVIII/vWF measurement, with no evidence of structural abnormality on multimeric analysis. Type 2 is usually characterised by the lack of large multimers and abnormally low levels of vWF:RCo in comparison with VWF:Ag. In this group, several subtypes, sometimes with recessive inheritance, have been observed. The phenotypes of type 2 is classified as A (patients with the absence of high molecular weight vWF-multimers and decreased platelet-dependent function), B (patients with hyperresponsiveness to ristocetin), M (patients with qualitative variants with decreased platelet-dependent function but with high molecular weight multimers present) and N (patients with defective FVIII-VWF binding). Type 3 is recessively transmitted and patients have very low or undetectable levels in all FVIII/vWF measurements.
vWF deficiency may also lead to phenotypical haemophilia A as vWF is essential for the function of FVIII. In these cases, the half-life of FVIII is decreased. Thus, patients suffering from vWD do often also suffer from FVIII deficiency. The reduced FVIII activity is not a result of a gene defect, but rather an indirect consequence of the vWF deviation. Normally, decreased ristocetin-cofactor activity and vWF antigen level are observed in vWD patients, whereas this is not the case in haemophilia A patients.
Haemophilia A is caused by partial or complete lack of the important coagulation protein FVIII, which circulates in the plasma completed with vWF, in the patient""s blood. The disease is inherited in a X-chromosome recessive pattern, and therefore it mainly affects males, but heterozygous females are asymptomatic carriers of the disease. The FVIII gene has been located to the long arm of the X-chromosome.
The coagulation process is an enzymatic cascade reaction enrolling intricate feedback activating and inactivating mechanisms. Most of the activated enzymes of the cascade require co-factors to be efficient, and some of these co-factors are plasma proteins which exist in both an active and an inactive form. Coagulation FVIII is one of such co-factors. In haemophilia A patients, a delayed clot formation is observed leading to excessive bleeding and poor wound healing. Haemorrhagic episodes in patients with haemophilia A can be managed by substituting FVIII.
The main symptoms of haemophilia A are excessive and prolonged bleeding. In severe haemophilia (less than 1% FVIII present), the most common sites of bleeding are in the large joints of the limps and in the large muscles. Unless such bleeding is controlled promptly by infusion of FVIII, the main chronic complications of haemophilia, namely arthropathy and muscle atrophy will occur. In mild haemophilia A (5-40% FVIII present), bleeding usually does not occur except after trauma. Moderately severe haemophilia A (1-5% FVIII present) has clinical features in between severe and mild haemophilia A and may show the largest individual variation.
In vWD, the main goal of treatment is to correct the bleeding time and FVIII defects. This is conventionally accomplished by administering the synthetic drug desmopressin (DDAVP) or by replacement therapy with FVIII/vWF preparations.
However, some patients with vWD do not respond to DDAVP treatment. Furthermore, patients initially responsive to DDAVP treatment may become refractory, when repeated infusions are given to maintain hemostasis over long periods of time. In these situations, the most commonly used treatment is replacement therapy with blood products such as cryoprecipitates or other concentrates prepared from plasma.
Cryoprecipitates are vWF-rich preparations obtained by thawing frozen blood plasma to 2-4xc2x0 C. and isolating the non-dissolved fraction. However, treatment with cryoprecipitates has been shown not to be sufficiently effective as correction of the bleeding time was only obtained in 24% of the cases, only partially in 36% of the cases, whereas no effect was observed in 40% of the cases (see Ref. 1). Furthermore, there is a high risk of transmitting life-threatening blood-borne viruses such as HIV and hepatitis viruses since virucidal methods cannot be applied directly to the cryoprecipitates normally used. Another disadvantage is the high content of fibrinogen and anti-A and anti-B agglutinins in these preparations, the presence of which may lead to hyper-fibrinogenaemia and anaphylactic reactions in patients with blood types A or B. Furthermore, due to the low concentration of vWF in this type of preparations, large volumes of fluid must be infused, and repeated infusions over time entail exposure to a large number of different donors thereby increasing the risk of infection with blood-borne viruses. These facts render the use of cryoprecipitates unsuitable for treating vWD.
In spite of the above-mentioned limitations, cryoprecipitates have been used in the treatment of patients suffering from haemophilia A. To avoid the above-mentioned problems, concentrates of FVIII, which are virus inactivated, have been developed. However, as the quality and safety of such concentrates have increased, the content and quality of vWF have diminished. High amounts of vWF are often present in low purity concentrates, whereas the content is low in medium and high-purity FVIII concentrates. In very high-purity concentrates such as affinity purified concentrates, vWF is almost absent. Thus, most high- and very high-purity concentrates are not suitable for treatment of vWD patients, whereas the low-purity concentrates contain high amounts of fibrinogen and high anti-A and anti-B agglutinin titres, thus entailing the above-mentioned disadvantages.
It has been found that a high content of high-molecular weight vWF (HMW-vWF)-multimers is essential for the effectiveness of the vWF preparation. Conventionally used preparations lack such HMW-vWF-multimers. This could be due to the presence of platelet proteases degrading the HMW-multimers during fractionation. As described in U.S. pat. No. 4,710,381 A (Ref. 2), it is important removing blood platelets from plasma intended for fractionation thereby hindering calcium-activated protease(s) present in blood platelets from being released and cause degradation of vWF, or, alternatively, adding calcium-chelating agents during fractionation thereby hindering released protease(s) from being calcium-activated.
A marked degree of vWF-fragmentation has been demonstrated in cryoprecipitates prepared from pooled plasmapheresis plasma, which is the starting fraction for producing most commercial concentrates (see Ref. 3 and Ref. 4). The vWF-degradation was shown to be proportional to the number of platelets present in the starting plasma. The degradation could be greatly diminished when collecting plasma in the presence of protease inhibitors (e.g. EDTA). On the basis of these findings, it was concluded that the loss of HMW-multimers resulted from the use of large plasma pools containing units of poorly centrifuged (plasmapheresis) plasma with an excessive number of residual platelets and leukocytes, lysing and liberating vWF-degrading proteases when the plasma is frozen and thawed. It was further concluded that vWF-preparations which had an intact HMW-vWF-multimeric pattern could not be prepared from pooled plasma containing poorly centrifuged plasmapheresis or whole blood plasma which had been frozen (see Ref. 3 and 4).
As outlined above, the known purification procedures result in loss of essential HMW-multimers. As none of the currently available preparations are, at the same time, sufficiently effective, pure and virus safe, there is a strong need for a preparation which at the same time is safe for the patient, effective, easily prepared and economically feasible.
These goals are fulfilled by the preparation of the present invention.
In EP 469 985 B1 (Ref. 5), a process for manufacturing vWF of high purity, largely devoid of FVIII:C is disclosed, which process comprises purification by ion exchange chromatography using a first chromatography column onto which FVIII:C is adsorbed. The non-retained fraction which contains the vWF is then treated so as to decrease the ionic strength, and the vWF fraction is then subjected to ion exchange chromatography, whereby vWF is adsorbed onto the gel of a second column with a first elution solution and then desorbed using a second elution solution of higher ionic strength than the first elution solution yielding the high purity vWF product. The second ion change chromatography is performed using a gel for exchanging anionic type ions. The crude solution containing FVIII:C as well as vWF may be subjected to diafiltration prior to the first purification step. In EP 469 985 B1, it has not been suggested to use gel filtration nor to use cation exchange chromatography for purification.
In WO 91/07438 A1 (Ref. 6), a method for isolating FVIII from other proteins using gel filtration is described. The gel filtration medium is constituted of particles being inert to FVIII. The method has not been used for preparing vWF preparations.
EP 705 846 A1 (Ref. 7), WO 96/10584 A1 (Ref. 8) and WO 97/34790 A1 (Ref. 9), respectively, describe methods for purifying vWF or the FVIII:C/vWF complex using anion exchange chromatography and affinity chromatography on immobilised heparin. High purity vWF products are obtained from both plasma-derived starting materials and cell supernatants containing recombinant vWF. The content of fibrinogen and fibronectin has not been measured or specified in any of the vWF products, and the multimeric structure has not been characterised using agarose gels that are suitable for separating HMW-multimers composed of 13 or more promoters. Furthermore, the gel electrophoresis methods used for evaluating the structural integrity of the vWF-multimers indicate that the products have a very low content of such HMW-multimers.
In EP 705 846 A1, a process for separating vWF into HMW-vWF and LMW-vWF is described, by which process vWF is bound to immobilised heparin, and the HMW-vWF and LMW-VWF are eluted at different salt concentrations. The affinity chromatography on immobilised heparin may be carried out at pH 6.0% to 8.5, preferably at pH 7.4. The HMW-VWF is eluted at a higher salt concentration than the salt concentration used for eluting LMW-vWF. Furthermore, a HMW-vWF fraction and a LMW-VWF fraction are claimed. Whereas the LMW-vWF fraction is claimed to contain at least 83% dimers, at maximum 16% tetramers and at maximum 1% other multimers, no such claims have been made in the case of the HMW-vWF fraction. A characterisation of the multimeric structure of a recombinant HMW-vWF fraction obtained by the process shows that the content of multimers composed of 13 or more protomers is less than 1%. The platelet aggregation activity of the fraction is specified to be at least 50% improved compared to the activity of vWF in the mixture of LMW-vWF and HMW-VWF. In Examples 5 and 6, recombinant vWF is purified using a combination of anion exchange chromatography and affinity chromatography using immobilised heparin.
In WO 96/10584 A1, a process for extracting high-purity vWF is described, whereby recombinant vWF is subjected to anion exchange chromatography using a quaternary amino-type anion exchanger. The-recombinant vWF is bound at a salt concentration below 270 mM and eluted at a salt concentration above 270 mM. The so obtained fraction is preferably purified further using affinity chromatography on immobilised heparin. Again, the obtained vWF has not been characterised as it is only stated that the vWF fraction contains multimers having a high structural integrity.
From WO 97/34930 A1, a stabile FVIII/vWF-complex is known, which complex contains HMW-vWF-multimets and which further does not contain LMW-VWF molecules and proteolytic vWF decomposition products. It is further stated that the complex has a specific platelet agglutination activity of at least 50 IU/mg vWF:Ag. The FVIII/vWF complex is obtained using affinity chromatography on immobilised heparin, and the high stability is obtained by introducing a washing buffer containing 10 mM CaCl2 during the anion exchange step. This washing step ensures the removal of proteolytic vWF decomposition products as well as some proteolytic proteins. Again, the multimeric structure has not been characterised using suitable agarose gels able to separate HMW-multimers composed of 13 or more protomers, but the gel electrophoresis methods used for evaluating the structural integrity of the vWF-multimers indicate that the products have a very low content of such multimers.
In accordance with the claims, the present invention relates to a vWF preparation comprising HMW-vWF-multimers, a process for preparing vWF-containing preparations as well as to the use of such preparations for preparing a medicament for the treatment of bleeding disorders. Furthermore, the invention relates to a pharmaceutical composition comprising said preparation and to a method for treating bleeding disorders.
Thus, in the broadest aspect, the present invention relates to a high-purity vWF-containing preparation, comprising at least 15% vWF-multimers composed of 13 or more protomers calculated on the basis of the total content of vWF-multimers and further having a low content of fibrinogen and fibronectin.
The following abbreviations are used throughout the present context.
As used herein, the term xe2x80x9cLMW (low molecular weight)-vWF mulitimersxe2x80x9d is intended to mean multimers composed of one to six protomers and having a molecular weight of from approximately 500 kD to approximately 3,000 kD, the term xe2x80x9cIMW (intermediate molecular weight)-vWF-multimersxe2x80x9d is intended to mean multimers composed of seven protomers to twelve protomers and having a molecular weight of from approximately 3,500 kD to approximately 6,000 kD, and the term xe2x80x9cHMW (high molecular weight)-vWF-multimersxe2x80x9d is intended to mean multimers composed of thirteen or more protomers and having a molecular weight of approximately 6,500 kD and higher.
It is a generally accepted fact that purification of a vWF-containing preparation having intact HMW-multimeric pattern and being prepared from pooled frozen plasmapheresis plasma cannot be obtained without, at the same time, the use of protease inhibitors being required. In accordance with the present invention, it has been demonstrated that a high-purity vWF-containing preparation containing practically all the HMW-vWF-multimers present in plasma can, surprisingly, be produced from normal pooled plasma, in particular from plasmapheresis plasma, without rendering necessary the use of protease inhibitors. The preparations can furthermore be prepared from plasma, which has been frozen and thawed.
High-purity plasma preparations are often combined with the stabilising protein human serum albumin which is regarded as completely safe and harmless. However, addition of albumin does give rise to difficulties in assessing the purity or specific activity of a preparation as the specific activity is traditionally expressed as the content of the active ingredient (e.g. vWF) relative to the total protein content. Thus, the total amount of quantitated protein in albumin-stabilised preparations will include the co-purified contaminants of concern as well as added albumin stabiliser.
The specific activity of a preparation should be assessed relative to specific contaminating proteins like fibrinogen and fibronectin. In most FVIII/vWF-preparations, fibrinogen and fibronectin are the main contaminating substances due to their cold-insoluble characteristics because of which they are precipitated and collected together with FVIII/vWF during cryoprecipitation which as earlier stated is the initial step of traditional plasma fractionation.
The result obtained by vWF-multimer analyses depends very much on the exact assay conditions, especially the percentage of agarose present in the gel matrix. Thus, the presence or absence of HMW-vWF-multimers can only be determined in gels of low agarose concentration (see Ref. 10). Accordingly, if the vWF-multimer analysis gel matrix contains acrylamide, it is important to keep the acrylamide concentration low when detecting HMW-vWF-multimers. Ruggeri and Zimmerman (see Ref. 11) showed that HMW-vWF-multimers cannot enter gels consisting of 2.5% acrylamide and 0.8% agarose or gels consisting of 3.0% acrylamide and 0.5% agarose, and are therefore not detectable in these gels whereas they do enter gels consisting of 0.8% agarose and 1.75% acrylamide. If this important impact of the gel composition on the vWF-multimer assessment is not considered, conflicting conclusions of vWF-multimer analyses might be drawn.
Accordingly, only low agarose and acrylamide concentrations in the gels are suitable for detecting HMW-vWF-multimers as positive vWF-multimer results obtained in higher concentration gels might be incorrect. Therefore, the analysis conditions (especially the gel composition) used for detecting HMW-vWF-multimers need to be specified in order to evaluate and compare results obtained.